Neurturin (NTN) is a recently characterized member of the glial cell line-derived neurotrophic factor (GDNF)-family which, like GDNF, can promote the survival of certain populations of neuronal cells in peripheral and central nervous systems. To elucidate the roles of NTN and a novel glycosyl-phosphatidylinositol (GPI)-linked receptor protein GFRalpha-3, a member of GDNF-family receptor alpha, in the regulation of peripheral trigeminal innervation and tooth formation, their expression patterns during mouse embryonic (E) and early postnatal (P) development (E10-P5) of the first branchial arch were analyzed by in situ hybridization. NTN mRNAs were observed in oral and cutaneous epithelia of the mandibular process at all studied stages and expression became gradually restricted to the suprabasal epithelial cells. In addition, transcripts were also detected in the epithelium of whisker follicles. In the developing first molar tooth germ, NTN showed a developmentally regulated, spatiotemporally changing expression pattern, which partially correlated with the development of innervation. During the initiation of tooth formation NTN mRNAs were expressed in dental epithelium and during later embryonic development transcripts appeared in the dental papilla mesenchyme. In addition, some transcripts were seen in the dental follicle. During postnatal development, NTN expression was restricted to the dental follicle of the incisor tooth germs. GFRalpha-3 mRNAs were not detected in teeth, but an intense expression was seen in non-neuronal cells surrounding trigeminal nerve fibers and in the trigeminal ganglia during E11-E15. Ganglion explant cultures showed that trigeminal neurons start to respond to exogenous NTN at E12, which correlates to the earlier reported appearance of the Ret-tyrosine kinase receptor in the trigeminal ganglion. Local application of NTN with beads on isolated dental mesenchyme did not stimulate cell proliferation or prevent apoptotic cell death. In addition, exogenous NTN had no effects on tooth morphogenesis in in vitro cultures. Taken together, because trigeminal neurons respond to NTN after first axons have reached their primary epithelial target fields, NTN is apparently not involved in the guidance of pioneer trigeminal nerves to their peripheral targets. However, our results show that NTN is a potent neuritogenic factor and, therefore, may act as a target-field-derived neurotrophic factor for trigeminal nerves during innervation of the cutaneous and oral epithelia as well as dental follicle surrounding the developing tooth. In addition, although NTN appears not to be directly involved in the regulation of tooth morphogenesis, it may have non-neuronal, organogenetic functions during tooth formation.